A single nucleotide polymorphism (SNP) is a genetic variation in a DNA sequence that occurs when a single nucleotide is replaced by one of the other three nucleotides. It results in differences between individuals, such as pathogenic causes or responses to therapeutic drugs. The detection and identification of single nucleotide polymorphisms has received a lot of attention, because it is linked not only to personalized medicines, but also to new drug development.
For rapid detection of single nucleotide polymorphisms, various detection methods based on real-time PCR technology have been used. Typical examples of these detection methods include assays using DNA intercalating fluorescent dyes, assays using DNA probes, and assays using PNA probes. However these methods have shortcomings in that the use of DNA intercalating fluorescent dyes is limited and the use of a program for analyzing melting curves is required (Kirk M. Ririe et al., Analytical Biochemistry 245:154, 1997; U. Hladnik et al., Clin Exp Med, 2:105, 2002).
DNA polymerase with 3′→5′ proofreading activity ensures high fidelity in DNA replication (Drake, J. W. et. al., Cold Spring Harb. Symp. Quant. Biol., 33:339, 1968; Drake, J. W. et. al., Nature, 221:1128, 1968; Goodman, M. F. et. al. Genetics, 148:1475, 1998), and many proofreading polymerases with 3′ exonuclease activity were been found.
DNA polymerase with proofreading activity ensures high fidelity in DNA replication in vivo, but when DNA polymerase with proofreading activity is applied to a polymerase chain reaction with conventional allele-specific primers, there is a problem in that, because a mismatched base at the 3′ end is removed, the end of the primer is extended regardless of complete hybridization or incomplete hybridization of the primer with DNA used as a template (Zhang, J. et al., Mol. Biotechnol., 24:105, 2003).
Due to this problem, activity of DNA polymerase with proofreading was hardly used in studies on the detection of mutations. However, in recent years, there have been studies on a method of detecting a mutation in a target nucleic acid using a method of modifying the 3′ end of primers, such as a method of labeling the 3′ end of the primers or conjugating a 3′ exonuclease-resistant factor to the 3′ end, or a method of removing the —OH group of the nucleotide at the 3′ end or replacing the —OH group with other residue (Zhang, J. et al., Current Drug Disc., 9:21, 2001; Bi, W. L. and Sambrook, P. J., Nucleic Acids Res., 26:3073, 1998).
For example, in the case in which the 3′ end of a primer is labeled, when the primer binds complementarily to the template DNA, a final amplification product is produced while the label at the 3′ end is maintained, but when the primer mismatches the template DNA, the label at 3′ end is removed by the proofreading activity of DNA polymerase with proofreading, and thus a final amplification product free of the label is produced, indicating that a mutation can be detected on the presence or absence of the label. Based on this principle, allele-specific primers having 3′ ends modified in various ways, and DNA polymerase with proofreading activity, can be applied to various platforms, including real-time PCR, multi-well plate and microarray techniques.
Accordingly, the present inventors have made extensive efforts to detect a target nucleic acid using a 3′ end-modified primer and a DNA polymerase having proofreading activity, and as a result, have found that a target nucleic acid is specifically amplified using an allele-specific reactive primer (ASRP) in which a nucleotide at the 3′ end is modified, in the presence of a DNA polymerase having proofreading activity, thereby completing the present invention.